CN106635931B - Engineering bacteria for BOD biosensor - Google Patents
Engineering bacteria for BOD biosensor Download PDFInfo
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- CN106635931B CN106635931B CN201710086987.7A CN201710086987A CN106635931B CN 106635931 B CN106635931 B CN 106635931B CN 201710086987 A CN201710086987 A CN 201710086987A CN 106635931 B CN106635931 B CN 106635931B
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- engineering bacteria
- bod
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- bod biosensor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/1806—Water biological or chemical oxygen demand (BOD or COD)
Abstract
The present invention proposes a kind of engineering bacteria for BOD biosensor, belongs to water quality monitoring field, and the engineering bacteria adaptive surface is wider, active high, stability and favorable reproducibility, can satisfy different types of water sample detection demand.The engineering bacteria is mixed by bafillus natto Bacillus natto and pseudomonad Pseudomonas sp. with weight ratio 2:1.The present invention can satisfy different types of water sample detection demand when quickly measuring applied to BOD biosensor.
Description
Technical field
The invention belongs to water quality monitoring field more particularly to a kind of engineering bacterias for BOD biosensor.
Background technique
Biochemical oxygen demand (BOD) (Biochemical Oxygen Demand, BOD) is the organic matter monitored in water body, especially
One important parameter of biodegradable content of organics is one of most common water body organic contamination overall target.
The standard determination method of BOD is method on the five, i.e., sample is cultivated 5 days in 20 ± 1 DEG C of temperature, and measurement culture front and back is molten respectively
Oxygen is solved, the difference between the two is 5 days biochemical oxygen demand (BOD)s, is indicated with BOD5.But this method operation is more complex, and time-consuming, as a result quasi-
Exactness and poor reproducibility are unable to satisfy the requirement quickly measured in current environment monitoring.
Quickly, the various Complex water body BOD values of Accurate Determining are the Main ways of biochemical oxygen demand (BOD) research, therefore it is required that sensing
Device has response to biodegradable organic matters various in water sample, while to be comparable with conventional BOD test method.But
The shortcomings that based on above-mentioned detection method so that how to concentrate research hotspot in the excellent selection of biological sensitive materials and efficiently it is micro-
The development of biomembrane, can quickly, BOD value in Accurate Determining water body, realize on-line continuous measurement, be adapted to modern environment
Monitoring and management requirement by be this field research important topic.
Summary of the invention
The invention proposes a kind of engineering bacteria for BOD biosensor, adaptive surface is wider, active high, stability and
Favorable reproducibility can satisfy different types of water sample detection demand.
In order to achieve the above object, the technical solution adopted by the present invention are as follows:
The present invention provides a kind of engineering bacterias for BOD biosensor, by bafillus natto Bacillus
Natto and pseudomonad Pseudomonas sp. are mixed with weight ratio 2:1.
As optimal technical scheme, the bafillus natto Bacillus natto selects stationary phase cells, the vacation
Monad Pseudomonas sp. selects logarithmic phase cell.
As optimal technical scheme, the cultivation temperature of the bafillus natto Bacillus natto is 33 DEG C, described
The cultivation temperature of pseudomonad Pseudomonas sp. is 25 DEG C.
The present invention also provides a kind of microbial films for BOD biosensor, using such as any of the above-described technical side
Engineering bacteria described in case for BOD biosensor is as strain.
It further include embedding medium as optimal technical scheme, the weight ratio of the embedding medium and the engineering bacteria is 1:1.
As optimal technical scheme, the embedding medium is sodium alginate.
The present invention also provides a kind of microbe immobilized particles for BOD biosensor, using such as any of the above-described
Item technical solution is used for the engineering bacteria of BOD biosensor as filler.
Compared with prior art, the advantages and positive effects of the present invention are:
Engineering bacteria provided by the present invention for BOD biosensor is mixed bacteria, rather than single culture or straight
It connects using activated sludge, this, which not only solves single culture, can only adapt to the defect of single water quality, also can avoid dirty using activity
The unstability and uncertainty measured when mud is as sensitive material.The mixed bacteria adaptive surface is wider, active high, stability and
Favorable reproducibility, not only applicable measurement standard BOD sample, can also meet the detection of different types of organic matter and waste water type
Demand.Above-mentioned mixed bacteria is used in BOD biosensor can greatly to improve detection speed, it can be complete in 8-30 minutes
At coherent detection.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
The embodiment of the invention provides a kind of engineering bacterias for BOD biosensor, by bafillus natto
Bacillus natto and pseudomonad Pseudomonas sp. are mixed with weight ratio 2:1.
Above mentioned embodiment provide a kind of engineering bacteria, which is the mixed bacteria of said two devices strain, uses mixing
Strain not only can avoid the defect that can only adapt to single water quality that single culture occurs, can also show adaptation as engineering bacteria
Face is wider, active high, stability and favorable reproducibility, and can meet the detection need of different types of organic matter and waste water type
The advantage asked.In the present embodiment, the weight ratio of the two is specifically defined, here mainly in view of in institute of the embodiment of the present invention
The engineering bacteria of offer is the engineering bacteria quickly measured for BOD biosensor, and usage amount is few and few (dry thin when detecting
Born of the same parents' use is mg grades), but need while the detection demand for meeting different quality, reach high detection precision requirement.
Therefore, in order to reach above-mentioned desired effect, the embodiment of the present invention also optimizes the selection of strain and the proportion relation of the two.It can
With understanding, the selection of above-mentioned strain can also be extended for the homologous or homologous series strain of two strains respectively, but it is found through experiment that,
The engineering bacteria mixed by homologous or homologous series strain is since the similitude of cyclic DNA and metabolic mechanism is higher, class of degrading
Type difference very little, different degrees of there are multi-party planar defects, can not effectively achieve the desired results.
In a preferred embodiment, the bafillus natto Bacillus natto selects stationary phase cells, the vacation
Monad Pseudomonas sp. selects logarithmic phase cell.In the present embodiment, the preferred bacterium cell of different times, this
The mainly growth stability in view of the two and mixed stability, so that the stability and reproducibility of the mixed bacteria
Preferably.
In a preferred embodiment, the cultivation temperature of the bafillus natto Bacillus natto is 33 DEG C, described
The cultivation temperature of pseudomonad Pseudomonas sp. is 25 DEG C.In the present embodiment, specific cultivation temperature has been advanced optimized
Under above two strain, this be mainly also consider strain performance play.Strain under above-mentioned cultivation temperature is mixed
Engineering bacteria is made, is more advantageous to the stability and reproducibility of the mixed bacteria.
The embodiment of the invention also provides a kind of microbial films for BOD biosensor, using such as any of the above-described
Engineering bacteria described in embodiment for BOD biosensor is as strain.The embodiment of the present invention provides microbial film by institute
The engineering bacteria used can solve existing BOD biosensor microbial film for mixed bacteria provided by the above embodiment
The disadvantages of adaptability is relatively narrow, performance is unstable, biological fixation is poor.
It further include embedding medium in an alternative embodiment, the weight ratio of the embedding medium and the engineering bacteria is 1:1.?
It lists in the present embodiment and is grouped as in the middle part of microbial film, it is to be understood that the composition of microbial film is not limited to above-mentioned
Embedding medium may also include ventilated membrane etc. in a kind of optional microbial film.In order to reach good air permeability effect, ventilated membrane
Aperture can be 0.2-0.7 μm, can be cellulose acetate film, nitrocellulose membrane, any one in GF-C film.In an optional system
During standby, ventilated membrane can be bonded in the two sides of double-sided adhesive, by punching on double-sided adhesive, then by engineering bacteria and embedding medium
It is uniformly mixed and is coated on ventilated membrane corresponding to the circular hole of double-sided adhesive and microbial film is made.In order to reach good embedding effect
The weight ratio of fruit, embedding medium and engineering bacteria can be 1:1.In an alternative embodiment, the embedding medium is sodium alginate.It can manage
It solves, the embedding medium in the present embodiment is not limited to sodium alginate, but with sodium alginate and engineering bacteria provided in this embodiment
Cooperate embedding effect achieved best.
The embodiment of the invention also provides a kind of microbe immobilized particles for BOD biosensor, using as above
Any one embodiment is stated for the engineering bacteria of BOD biosensor as filler.Microorganism provided in an embodiment of the present invention is fixed
Change particle since used filler is mixed bacteria provided by the above embodiment, not only conforming to property is strong, performance is stable, raw
Object stationarity is good, and microsyringe sample introduction can be used, and application surface is wider, and due to micro- in each immobilization particle
Biological content is controllable, thus the measurement of BOD biosensor can also be made relatively stable.
The engineering bacteria that BOD biosensor is used for provided by the embodiment of the present invention is introduced in detail in order to become apparent from, under
Face will be described in conjunction with specific embodiments.
Embodiment 1
A kind of microbial film for BOD biosensor, the microbial film include ventilated membrane, engineering bacteria, embedding medium,
Double-sided adhesive etc., in which:
The aperture of ventilated membrane is 0.2-0.7 μm (preferably 0.45 μm), in cellulose acetate film, nitrocellulose membrane, GF-C film
Any one；
Engineering bacteria is by bafillus natto Bacillus natto and pseudomonad Pseudomonas sp. with weight ratio 2:
1 mixes；
Embedding medium is sodium alginate, and the weight ratio with engineering bacteria is 1:1；
It is punched on double-sided adhesive every 4cm, aperture (0.5-0.8cm, preferably 0.6cm), ventilated membrane is bonded in the double-sided adhesive
Two sides, engineering bacteria and embedding medium are coated on after mixing on ventilated membrane corresponding to the circular hole of the double-sided adhesive.
Engineering bacteria the preparation method is as follows:
Laboratory apparatus: electronic balance, glass bar, conical flask, conical flask, electric furnace, high-pressure sterilizing pot, test tube, connects beaker
Kind ring, alcolhol burner, aseptic operating platform, biochemical cultivation case, shaking table, centrifuge, centrifuge tube (10ml), liquid-transfering gun
Experimental material: sewage sample (from different sewage treatment plant and soil), 0.8% physiological saline,
Basal medium: beef extract 0.75g, peptone 1.5g, sodium chloride 0.75g, agar 3g,
Phosphate buffer solution: (Na is weighed2HPO4·12H2O) 1.79 grams of disodium hydrogen phosphate, weigh (KH2PO4) di(2-ethylhexyl)phosphate
0.68 gram of hydrogen potassium, constant volume is in 1000ml distilled water up to 0.005molL-1Phosphate buffer solution, pH=7.0,
Pure bacterial strain is expanded culture with fluid nutrient medium, and concussion 48h (presses 33 DEG C or 25 DEG C of required temperature, 140rpm),
Later centrifugation (4000rpm, 6min) obtain engineering bacteria thallus, with 0.8% physiological saline elution three times, concussion for 24 hours (30 DEG C,
140rpm), centrifugation obtains engineering bacteria.
Performance test
Embodiment 1 is used in the practical measurement of different water samples by the microbial film that mixed bacteria is prepared.Further,
It is obtained with by single culture (trichosporon cutaneum that national sector standard (HJ/T86-2002) is recommended to use) and active sludge treatment
To microbial film compare, it is as follows in conjunction with traditional method evaluation of result on the 5th, referring specifically to table 1.
1 evaluation result of table
Note: deviation is microbial film measuring method and method contrast difference on the 5th in table
The result shows that: the biomembrane that the mixed bacteria as provided by the embodiment of the present invention is prepared not only stablize by performance
Property, favorable reproducibility, but also the detection demand of different types of organic matter and waste water type can be met.Compared to by single bacterium
For microbial film and tradition the method result on the 5th that kind and active sludge treatment obtain, relative deviation is respectively less than 5%, for not
There is good degradability with pollutant.
Claims (6)
1. a kind of engineering bacteria for BOD biosensor, which is characterized in that by bafillus natto Bacillus natto and
Pseudomonad Pseudomonas sp. is mixed with weight ratio 2:1；The bafillus natto Bacillus natto is selected
Stationary phase cells, the pseudomonad Pseudomonas sp. select logarithmic phase cell.
2. the engineering bacteria according to claim 1 for BOD biosensor, which is characterized in that the natto gemma bar
The cultivation temperature of bacterium Bacillus natto is 33 DEG C, and the cultivation temperature of the pseudomonad Pseudomonas sp. is 25 DEG C.
3. a kind of microbial film for BOD biosensor, which is characterized in that use and be used for as claimed in claim 1 or 2
The engineering bacteria of BOD biosensor is as strain.
4. the microbial film according to claim 3 for BOD biosensor, which is characterized in that it further include embedding medium,
The weight ratio of the embedding medium and the engineering bacteria is 1:1.
5. the microbial film according to claim 4 for BOD biosensor, which is characterized in that the embedding medium is
Sodium alginate.
6. a kind of microbe immobilized particles for BOD biosensor, which is characterized in that using such as claims 1 or 2 institute
The engineering bacteria for BOD biosensor stated is as filler.
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| CN201710086987.7A CN106635931B (en) | 2017-02-17 | 2017-02-17 | Engineering bacteria for BOD biosensor |
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| CN201710086987.7A CN106635931B (en) | 2017-02-17 | 2017-02-17 | Engineering bacteria for BOD biosensor |
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| CN106635931A CN106635931A (en) | 2017-05-10 |
| CN106635931B true CN106635931B (en) | 2019-09-27 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1186115A (en) * | 1996-12-25 | 1998-07-01 | 中国科学院长春应用化学研究所 | Method for biological sensor produced by latex embedding enzyme |
| CN1563354A (en) * | 2004-03-24 | 2005-01-12 | 河北科技大学 | Strain in use sensor of measuring oxygen quantity needed by industrial wastewater or seawater biochemistry-method for cultivating bacillus licheniformis |
| CN103773753A (en) * | 2013-12-31 | 2014-05-07 | 青岛万源清环保技术有限公司 | Microbial film for biosensor and preparation method thereof |
| CN103843184A (en) * | 2011-06-14 | 2014-06-04 | 凯博瑞创新公司 | Biological oxygen demand sensors |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100555840B1 (en) * | 2004-05-31 | 2006-03-03 | 장덕진 | A biosensor for detecting inhibitors of ammonia oxidation |
| WO2006075030A2 (en) * | 2005-01-17 | 2006-07-20 | Universidad Técnica Federico Santa María | Biosensor for determining the biochemical oxygen demand (bod) by respirometry |
-
2017
- 2017-02-17 CN CN201710086987.7A patent/CN106635931B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1186115A (en) * | 1996-12-25 | 1998-07-01 | 中国科学院长春应用化学研究所 | Method for biological sensor produced by latex embedding enzyme |
| CN1563354A (en) * | 2004-03-24 | 2005-01-12 | 河北科技大学 | Strain in use sensor of measuring oxygen quantity needed by industrial wastewater or seawater biochemistry-method for cultivating bacillus licheniformis |
| CN103843184A (en) * | 2011-06-14 | 2014-06-04 | 凯博瑞创新公司 | Biological oxygen demand sensors |
| CN103773753A (en) * | 2013-12-31 | 2014-05-07 | 青岛万源清环保技术有限公司 | Microbial film for biosensor and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 微生物传感器测定水中BOD的研究进展;张国伟 等;《环境监控与预警》;20101031;第2卷(第5期);第15-17、22页 * |
| 耗氧微生物的种类对光化学BOD传感膜性能影响的研究;林玲 等;《海洋技术》;20041231;第23卷(第4期);第53-57页 * |
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